Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2.

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.

History and current status of robotic totally endoscopic coronary artery bypass.

Robotic totally endoscopic coronary artery bypass (TECAB) is a minimally invasive endoscopic surgical approach using the daVinci robotic telemanipulation system to perform coronary artery bypass grafting on the arrested or beating heart. It is a procedure that can be a useful alternative to the classic open procedure performed through sternotomy. After extensive modeling in cadavers, the first clinical case was performed in June 1998 placing a left internal thoracic artery graft (LITA) to the left anterior descending artery completely robotically on the arrested heart. During the early and late 2000s, international groups have adopted this evolving technology, which has included iterations such as beating-heart TECAB, use of bilateral ITA grafting and radial artery grafting, as well as 3- and 4-vessel TECAB. TECAB is combined with percutaneous coronary intervention in hybrid procedures. Despite increasing complexity of endoscopic coronary bypass surgery, conversion rates to open bypass surgery have dropped significantly and operative times have decreased. Published major morbidities and mortality rates in arrested-and beating-heart TECAB have been cumulatively in the 0-2% range and are considered well within the expected range for these highly complex surgical procedures. Long-term survival and freedom from major adverse events also meet the standards of open bypass surgery.

Left-right patterning in the mouse requires Epb4.1l5-dependent morphogenesis of the node and midline.

The mouse node is a transient early embryonic structure that is required for left-right asymmetry and for generation of the axial midline, which patterns neural and mesodermal tissues. The node is a shallow teardrop-shaped pit that sits at the distal tip of the early headfold (e7.75) embryo. The shape of the node is believed to be important for generation of the coherent leftward fluid flow required for initiation of left-right asymmetry, but little is known about the morphogenesis of the node. Here we show that the FERM domain protein Lulu/Epb4.1l5 is required for left-right asymmetry in the early mouse embryo. Unlike other genes previously shown to be required for left-right asymmetry in the mouse, lulu is not required for specification of node cell identity, for Nodal signaling in the node or for ciliogenesis. Instead, lulu is required for proper morphogenesis of the node and midline. The precursors of the wild-type node undergo a series of rapid morphological transitions. First, node precursors arise from an epithelial-to-mesenchymal transition at the anterior primitive streak. While in the mesenchymal layer, the node precursors form several ciliated rosette-like clusters; they then rapidly undergo a mesenchymal-to-epithelial transition to insert into the outer, endodermal layer of the embryo. In lulu mutants, node precursor cells are specified and form clusters, but those clusters fail to coalesce to make a single continuous node epithelium. The data suggest that the assembly of the contiguous node epithelium from mesenchymal clusters requires a rapid reorganization of apical-basal polarity that depends on Lulu/Epb4.1l5.

Design and Characterization of a Quasi-Passive Pneumatic Foot-Ankle Prosthesis.

The majority of commercially available passive prosthetic feet are not capable of providing joint mechanics that match that of the intact human ankle. Due to their cantilever design, their stiffness characteristics contrast with what has been observed in the biological ankle, namely, an increase in stiffness during the stance phase of walking. In this paper, we introduce the design and control of a pneumatic foot-ankle prosthesis that attempts to provide biomimetic mechanics. The prosthesis is comprised of a pneumatic cylinder in series with a fiberglass leaf spring, and a solenoid valve to control the flow of air between the two sides of the cylinder. The solenoid valve acts as a mechanical clutch, enabling resetting of the ankle's equilibrium position. By adjusting the pressure inside the cylinder, the prosthesis can be customized to provide a range of ankle mechanics. A mechanical testing machine is used to compare the torque-angle curve of the pneumatic prosthesis with a low-profile passive prosthetic foot. Finally, data are presented of one transtibial amputee walking with the prosthesis at 1.2 m/s. The testing shows that the pneumatic prosthesis is capable of providing an appropriate range of motion as well a maximum torque of 94 Nm, while returning approximately 11.5 J of energy.

A mosaic genetic screen reveals distinct roles for trithorax and polycomb group genes in Drosophila eye development.

The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. We have used a mosaic genetic screen to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, we identified several members of the Polycomb and trithorax classes of genes encoding general transcriptional regulators. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors.

Depression, anxiety and neuropsychological test scores of candidates for coronary artery bypass graft surgery.

The effect of depression and anxiety upon neuropsychological test scores of candidates for coronary artery bypass graft (CABG) surgery was examined. Sixty patients were administered the Beck Depression Inventory II and the State-Trait Anxiety Inventory, along with a battery of neuropsychological tests. Regression analyses were conducted in which the neuropsychological test scores were predicted using age, education, depression, anxiety, and combined depression-anxiety scores. While age and education were significant predictors of several neuropsychological test measures, no significant regression analysis results were obtained for the depression, anxiety and combined depression-anxiety scores. Similarities and discrepancies between this research and previous studies are discussed. It appears that low levels of preoperative depression and anxiety states do not affect neuropsychological functioning among CABG candidates.

Benefits of off-pump bypass on neurologic and clinical morbidity: a prospective randomized trial.

BACKGROUND: Neurologic and clinical morbidity after coronary artery bypass grafting (CABG) can be significant. By avoiding cardiopulmonary bypass, off-pump CABG (OPCAB) may reduce morbidity. METHODS: Sixty patients (30 CABG and 30 OPCAB) were prospectively randomized. Neurocognitive testing was performed before the operation and 2 weeks and 1 year after the operation. Neurologic testing to detect stroke and (99m)Tc-HMPAO whole-brain single photon emission computed tomography scanning to assess cerebral perfusion were performed before the operation and 3 days afterward. Bilateral middle cerebral artery transcranial Doppler scanning was performed intraoperatively to detect cerebral microemboli. All examiners were blinded to treatment group. Clinical morbidity and costs were compared. RESULTS: Coronary artery bypass grafting was associated with more cerebral microemboli (575 +/- 278.5 CABG versus 16.0 +/- 19.5 OPCAB (median +/- semiinterquartile range) and significantly reduced cerebral perfusion after the operation to the bilateral occipital, cerebellar, precunei, thalami, and left temporal lobes (p < or = 0.01). Cerebral perfusion with OPCAB was unchanged. Compared with base line, OPCAB patients performed better on the Rey Auditory Verbal Learning Test (total and recognition scores) at both 2 weeks and at 1 year (p < or = 0.05), whereas CABG performance was statistically unchanged for all cognitive measures. Patients who underwent CABG had more chest tube drainage (1389 +/- 1256 mL CABG versus 789 +/- 586 mL OPCAB, p = 0.02) and required more blood (3.9 +/- 5.8 U CABG versus 1.2 +/- 2.2 U OPCAB, p = 0.02), fresh frozen plasma (3.0 +/- 6.0 U CABG versus 0.5 +/- 2.2 U OPCAB, p = 0.03), and hours of postoperative use of dopamine (16.3 +/- 21.2 hours CABG versus 7.3 +/- 9.7 hours OPCAB, p = 0.04). These differences culminated in higher costs for CABG ($23,053 +/- $5,320 CABG versus $17,780 +/- $4,390 OPCAB, p < 0.0001). One stroke occurred with CABG, compared with none with OPCAB (p = NS). One OPCAB patient died because of a pulmonary embolus (p = NS). CONCLUSIONS: Compared with CABG, OPCAB may reduce neurologic and clinical morbidity as well as cost.

One-stage triple hybrid arch debranching.

Traditionally, repair of complex arch and descending aortic disease has required a two-stage surgical approach. We describe a novel one-stage repair that combines primary revascularization of the subclavian with stent coverage of diffuse aortic disease. A 31-year-old woman with Marfan syndrome presented with a growing arch aneurysm. At age 16 years, she had undergone the Ross procedure followed by a redo sternotomy and valve conduit. On follow-up, a distal arch aneurysm measuring 6 cm with an aneurysmal upper thoracic aorta and a localized distal thoracic aortic dissection was identified. In one stage, triple hybrid off-pump arch debranching with endovascular stent grafting was performed using two GORE Hybrid Vascular Grafts to perform the distal left carotid and subclavian anastomoses, which provided complete primary arch debranching without requiring a carotid-subclavian shunt. The patient had no complications, was discharged home on postoperative day 3, and continues to do well 3 months postoperatively. We conclude that one-stage repair of complex aortic arch disease with primary revascularization of the subclavian is feasible.

Factors influencing hospital length of stay after robotic totally endoscopic coronary artery bypass grafting.

BACKGROUND: Robotic totally endoscopic coronary artery bypass grafting (TECAB) is an evolving minimally invasive technology with the potential to reduce hospital length of stay (LOS). Little is known about the factors that influence LOS after this procedure. The aim of this study is to define the preoperative, intraoperative, and postoperative variables that predict LOS after TECAB. METHODS: From 2001 to 2011, 541 patients, aged 60 years (range, 26 to 90 years), 394 (72.8%) male, 147 (27.1%) female, underwent TECAB using the daVinci telemanipulation system at one European and one American institution. Three hundred forty-six (63.9%) single-, 171 (31.6%) double-, 23 (4.2%) triple-, and 1 (0.2%) quadruple-vessel TECABs were carried out with an overall LOS of 6 days (range, 2 to 54 days) and 30-day mortality of 0.9% (5 of 541); 44.5% of patients (241 of 541) were hybrid intent-to-treat candidates. RESULTS: The following variables showed significant positive correlation with LOS: age, r = 0.188 (p < 0.001); Society of Thoracic Surgeons risk score, r = 0.263 (p < 0.001); EuroSCORE, r = 0.191 (p < 0.001); creatinine, r = 0.135 (p = 0.002); and operative time, r = 0.216 (p < 0.001). Other factors that had significant influence on LOS were hemodialysis (p = 0.037), cerebrovascular disease (p = 0.002), learning curve case (p < 0.001), intraoperative surgical problem (p < 0.001), conversion or on-table revision (p < 0.001), revision for bleeding (p < 0.001), postoperative stroke (p < 0.001), intraaortic balloon pump (p < 0.001), hemodialysis (p < 0.001), and atrial fibrillation (p < 0.001). By multivariate analysis, learning curve case, conversion or on-table revision, and revision for bleeding were independent predictors for prolonged LOS (defined as LOS > 6 days). CONCLUSIONS: Multiple variables affect LOS after TECAB. Older patients, patients on hemodialysis, patients with cerebrovascular disease, and those with higher general risk scores should expect prolonged LOS. Intraoperative surgical difficulties and conversion to open coronary artery bypass grafting also lead to extended LOS. Postoperative events that are known to prolong LOS in open coronary artery bypass grafting also prolong LOS after TECAB.

Robotically assisted hybrid coronary revascularization: does sequence of intervention matter?

OBJECTIVE: Hybrid coronary revascularization (HCR) is a treatment strategy for the revascularization of multivessel coronary disease that combines the advantages of both minimally invasive surgical techniques and percutaneous coronary intervention (PCI). The optimal sequence by which revascularization should be accomplished has not been determined. We investigated clinical outcomes in a series of patients planned for HCR via robotically assisted totally endoscopic coronary artery bypass (TECAB) and standard PCI based on revascularization sequence. METHODS: A total of 238 patients planned for HCR between 2001 and 2011 were divided into three groups based on treatment sequence: (a) TECAB before PCI, (b) PCI before TECAB, and (c) same-session procedure. Multiple procedural and clinical end points before discharge and up to 2 years after the procedure were compared between the three groups in an intention-to-treat analysis. Demographic features were reviewed to determine baseline differences between each group. RESULTS: Of the 238 patients, 175 (73.5%) underwent TECAB before PCI, 38 patients (16.0%) underwent PCI before TECAB, and 25 (10.5%) underwent a simultaneous revascularization procedure. At baseline, the patients undergoing TECAB before PCI were significantly older. There was a significantly higher incidence of previous myocardial infarction in the PCI-first group (P < 0.001). There was a significant difference in intensive care unit (ICU) length of stay (LOS), with shorter ICU stays in the simultaneous revascularization group (P = 0.031) and shorter hospital LOS in the PCI before TECAB group (P = 0.021). CONCLUSIONS: In conclusion, revascularization sequence did not dramatically impact clinical outcomes in our observational study. The patients undergoing PCI-first and same-session interventions had shorter hospital and ICU LOS compared with the patients undergoing surgery first. Our findings suggest that no revascularization approach is arbitrarily superior and that revascularization sequence should be individualized on the basis of patient presentation and anatomical considerations.

Robotic totally endoscopic multivessel coronary artery bypass grafting: procedure development, challenges, results.

Closed-chest totally endoscopic coronary artery bypass grafting (TECAB) is feasible using robotic technology. During the early phases, TECAB was restricted to single bypass grafts to the left anterior descending artery system. Because most patients referred for coronary artery bypass surgery have multivessel disease, development of endoscopic multiple bypass grafting is mandatory. Experimental work on multivessel TECAB was carried out in the early 2000s, and first clinical cases were already performed. With further technological development of operating robots, double, triple, and quadruple TECAB has become feasible both on the arrested heart and on the beating heart. To date, 161 cases of multivessel TECAB using the da Vinci telemanipulation systems are published in the literature. The main advances enabling multivessel TECAB were the availability of a robotic endostabilizer for beating heart procedures and increased surgeon skills using remote access heart-lung machine perfusion and endo-cardioplegia. Both internal mammary arteries can be harvested and both radial artery and vein graft can be used in multivessel TECAB. Y-grafting and sequential grafting are feasible. Multivessel endoscopic surgical revascularization can be combined with percutaneous coronary interventions in advanced hybrid coronary revascularization. Time requirements for multivessel TECAB are significant, and conversion rates to larger thoracic incisions are higher than those observed for single-vessel TECAB. Clinical short- and long-term outcomes, however, seem to meet the standards of open coronary bypass surgery through sternotomy. The main advantages of multivessel TECAB are a completely preserved sternum, use of double internal mammary artery even in risk groups, and a remarkably short recovery time.

Morphogenesis of the node and notochord: the cellular basis for the establishment and maintenance of left-right asymmetry in the mouse.

Establishment of left-right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop-shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi-step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left-sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left-right asymmetry.

The FERM protein Epb4.1l5 is required for organization of the neural plate and for the epithelial-mesenchymal transition at the primitive streak of the mouse embryo.

During early mouse development, a single-layered epithelium is transformed into the three germ layers that are the basis of the embryonic body plan. Here we describe an ENU-induced mutation, limulus (lulu), which disrupts gastrulation and the organization of all three embryonic germ layers. Positional cloning and analysis of additional alleles show that lulu is a null allele of the FERM-domain gene erythrocyte protein band 4.1-like 5 (Epb4.1l5). During gastrulation, some cells in lulu mutants are trapped in the primitive streak at an intermediate stage of the epithelial-mesenchymal transition; as a result, the embryos have very little paraxial mesoderm. Epithelial layers of the later lulu embryo are also disrupted: definitive endoderm is specified but does not form a gut tube, and the neural plate is broad and forms ectopic folds rather than closing to make the neural tube. In contrast to zebrafish and Drosophila, in which orthologs of Epb4.1l5 control the apical localization and activity of Crumbs proteins, mouse Crumbs proteins are localized normally to the apical surface of the lulu mutant epiblast and neural plate. However, the defects in both the lulu primitive streak and neural plate are associated with disruption of the normal organization of the actin cytoskeleton. We propose that mouse Lulu (Epb4.1l5) helps anchor the actin-myosin contractile machinery to the membrane to allow the dynamic rearrangements of epithelia that mediate embryonic morphogenesis.

Analysis of mouse embryonic patterning and morphogenesis by forward genetics.

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms.

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. The two genes are independently regulated by hyperplastic discs; decapentaplegic can still be misexpressed in cells mutant for both hyperplastic discs and hedgehog, and a repressor form of the transcription factor Cubitus interruptus can block decapentaplegic misexpression but not hedgehog misexpression. Loss of hyperplastic discs causes the accumulation of full-length Cubitus interruptus protein, but not of Smoothened, in both the eye and wing discs. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis.